Effect of different pretreatment on cryopreservation injury of native Cymbidium sinense rhizome tips

本實驗以台灣原生之報歲蘭（Cymbidium sinense （Andr.）Willd. ）為材料，主要目的在研究種子及根莖莖頂超低溫冷凍保存之方法，並探 討乾燥劑脫水、保護劑與不同濃度蔗糖培養基前處理對報歲蘭根莖莖頂細 胞膜系傷害之影響。同時配合穿透式電子顯微鏡觀察細胞微細構造之變化 ，以瞭解冷凍保存之處理過程對報歲蘭根莖莖頂影響之情形。 蘭花種 子移入冷凍管中，直接置入液態氮桶中進行超低溫冷凍保存，經液態氮冷 凍保存2年後仍能存活發芽。根莖莖頂先以乾燥劑脫水、保護劑、裹膠脫 水（encapsulation-dehydration）及不同濃度蔗糖培養基等前處理後， 進行超低溫冷凍保存結果均未存活，因此進一步測定乾燥劑脫水、保護劑 與不同濃度蔗糖培養基前處理後，膜脂質過氧化程度、離子滲漏及微細構 造之變化，以作為判斷冷凍保存處理過程之傷害依據。根莖莖頂未經液態 氮冷凍保存，僅以乾燥劑脫水、保護劑與不同濃度蔗糖培養基處理，莖頂 MDA生成量、離子滲漏程度皆已上升。穿透式電子顯微鏡觀察，發現經乾 燥劑脫水60分鐘後造成細胞質離、細胞質濃厚，但仍可見到粒線體，而細 胞壁開始變形；保護劑處理則造成細胞質離及產生小囊泡。以乾燥劑脫水 或PVS2冷凍保護劑處理根莖莖頂，再經冷凍保存後細胞膜附近皆有物質累 積，部份膜系產生破洞，顯示膜系構造已經受傷，此結果亦與冷凍保存後 高膜脂質過氧化及離子滲漏之結果相符合。 上述三種前處理結果以冷凍 保護劑之傷害最嚴重，故進行報歲蘭根莖莖頂冷凍保存應避免使用PVS2組 成之冷凍保護劑。由各項前處理傷害程度之結果獲知，以蔗糖培養基前處 理配合乾燥劑脫水60分鐘為限，可作為冷凍保存前處理模式，若能再進一 步探討相配合之其他處理或可獲致有效之冷凍保存技術。This study used native Cymbidium sinense as materials for investigation. The main purpose was to investigate the cryopreservation protocols of seeds and rhizome tips, and understand the degrees of damage on cell membrane after dehydration, cryoprotectant pretreatment or preculture in different sucrose concentrations of semi-solid culture medium. In the meanwhile, ultrastructural changes in rhizome tips were used to show the damage of cryopreservation procedure. Cymbidium sinense seeds were transferred to cryogenic vial, and immersed in liquid nitrogen for cryopreservation. The seeds still can germinate after cryopreservation for two years. There was no survival after the rhizome tips were pretreated with dehydration, cryoprotectant, encapsulation-dehydration or different sucrose concentrations of semi-solid culture medium, and then cryopreserved in liquid nitrogen. Furthermore, lipid peroxidation, ion leakage, and ultrastructural change were detected to judge the degree of damage on rhizome tips through cryopreservation procedure. The MDA production and ion leakage were all increased after treated with dehydration, cryoprotectant or different sucrose concentrations of semi-solid culture medium only without cryopreservation. The TEM ultrastructural examination revealed that the cell was plasmolyzed, cytoplasm concentrated and cell wall deformed, but mitochondria were still seen after dehydration for 60 mins. The cell was also plasmolyzed after cryoprotectant treatment but many vesicles appeared. Furthermore, cytosol was accumulated at the periphery of plasma membrane, and part of membrane systems was broken after dehydration and PVS2 pretreatment followed by cryopreservation. This result showed that membrane systems were damaged in accordance with the high lipid peroxidation and ion leakage detected after cryopreservation. The most serious injury of three pretreatments mentioned above was cryoprotectant pretreatment. Thus, the cryopreservation of rhizome tips of Cymbidium sinense should avoid using cryoprotectant composed with PVS2 ingredient. The result of this study suggests that pretreatment with sucrose semi-solid culture medium and dehydration with silica gel within 60 mins can be the basic procedure for cryopreservation of rhizome tips. To enhance the survival after freeze-thawing may need to combine additional treatment in the procedure